Pe cy7 conjugated anti mouse f4 80

Peritoneal cells were stained with a PE/Cy7-conjugated anti-mouse F4/80 (1:200, Cat#25-4801-82, eBioscience) and APC-eFluor780-conjugated anti-mouse CD11b (1:200, Cat#47-0112-82, eBioscience) for gating macrophages. PE/Cy7-conjugated anti-mouse F4/80 together with eFluor450 anti-mouse Ly6C (1:800, Cat#48-5932-80, eBioscience) was used to stain M1 phenotype macrophages and PE/Cy7-conjugated anti-mouse F4/80 together with FITC-conjugated anti-mouse CD206 (1:200, Cat#141704, Biolegend) was used to stain M2 phenotype macrophages as previously described (30 (link)). Briefly, cells were first blocked with anti-mouse CD16/CD32 (1:200, Cat#14-0161-81, eBioscience) to eliminate of Fc receptor-mediated antibody binding. Cell surface staining was performed by incubating cells with indicated antibodies for 15 min on ice. CD206 staining was carried out using the intracellular staining kit (Cat#00-5523-00, eBioscience) according to the manufacturer’s protocol. Cells were washed and analyzed on an LSR Fortessa flow cytometer (BD Biosciences, USA) using FlowJo software (BD Biosciences, USA).

Peritoneal macrophages from young (<3 months) and old (>18 months) mice were isolated and cultured as described previously. Macrophages were blocked with mouse BD Fc Block (BD Pharmingen, San Diego, CA) before double staining with PE-Cy7-conjugated anti-mouse F4/80 (1/100; 25–4,801, clone:BM8, eBiosciences) and anti-mouse IL10R1-PE (1/100; CD210, no. 559914, clone 1B1.3a, BD Biosciences, San Jose, CA) or rat IgG1,κ-PE isotype control (1/100; no. 554685, clone:R3–34, BD Biosciences). Acquisition was carried out using LSR-II cytometer (BD Biosciences), and the data were analysed using FlowJo Software (version 7.6.2).